Isolation and purification of Tartary buckwheat polysaccharides and their effect on gut microbiota

Abstract Tartary buckwheat (Fagopyrum tataricum) is rich in polysaccharides that can be utilized by the gut microbiota (GM) and provide several health benefits. However, the mechanisms underlying the action of these polysaccharides remain unclear to date. In this study, Tartary buckwheat polysaccharides (TBP) were purified, and five fractions were obtained. The composition of these fractions was determined using ion chromatography. Different TBP components were investigated regarding their probiotic effect on three species of Bifidobacteria and Lactobacillus rhamnosus. In addition, the effect of TBP on GM and short‐chain fatty acids (SCFAs) was evaluated. Results showed that the probiotic effect of TBP fraction was dependent on their composition. The polysaccharides present in different fractions had specific probiotic effects. TBP‐1.0, mainly composed of fucose, glucose, and d‐galactose, exhibited the strongest proliferation effect on L. rhamnosus, while TBP‐W, rich in glucose, d‐galactose, and fructose, had the best promoting effect on Bifidobacterium longum and Bifidobacterium adolescentis growth. Furthermore, TBP‐0.2, composed of d‐galacturonic acid, d‐galactose, xylose, and arabinose, exhibited its highest impact on Bifidobacterium breve growth. The composition of GM was significantly altered by adding TBP during fecal fermentation, with an increased relative abundance of Lactococcus, Phascolarctobacterium, Bacteroidetes, and Shigella. Simultaneously, the level of SCFA was also significantly increased by TBP. Our findings indicate that Tartary buckwheat can provide specific dietary polysaccharide sources to modulate and maintain GM diversity. They provide a basis for Tartary buckwheat commercial utilization for GM modulation.


| INTRODUC TI ON
Colonization of the gut microbiota (GM) in humans begins at birth and develops majorly in the first 3 years of life. It becomes more complex when children start eating solid food, gradually possessing microbiota roughly similar in structure and function to adults (Roswall et al., 2021). The GM composition in healthy adults is generally stable. However, when it is disturbed due to internal or external perturbations, the body often starts showing signs of various diseases. For instance, imbalanced GM in patients with type 2 diabetes decreased the number of common butyrate-producing bacteria, resulting in increased growth of various opportunistic pathogens (Gilbert et al., 2018;Qin et al., 2012).
Polysaccharides, as an essential component of the daily diet, can be involved in various physiological activities through the regulation of GM (Seedorf et al., 2014). Polysaccharides exert significant modulatory effects on host health by promoting the growth of certain probiotic bacteria, such as Lactobacilli and Bifidobacteria (Fernández et al., 2016), and inducing the expression of immunomodulatory and pathogen antagonistic molecules (Turroni et al., 2014).
Polysaccharides also promote the production of various types of short-chain fatty acids (SCFAs) that play a crucial role in host metabolism (Krautkramer et al., 2021). It was found that after 24 h of anaerobic fermentation of Porphyra haitanensis polysaccharides (PHP), the GM composition was remodeled due to the proliferation of probiotics and the inhibition of pathogens. The level of GM diversity was also significantly increased. In addition, the final concentrations of acetic acid, propionic acid, butyric acid, and total SCFAs were increased (Xu et al., 2019). Recently, polysaccharides' physiological functions were found to be closely related to their structure in terms of monosaccharide composition, relative molecular mass, molecular shape, and chain conformation (Yang et al., 2022). It is also known that polysaccharides from different plant sources provide a rich and diverse carbon source for the GM and are crucial components in maintaining intestinal homeostasis (Tannock, 2020). Moreover, understanding the relationship between plant polysaccharides composition and the regulation of microbial activity provides a basis for adopting a precise diet in routine.
Tartary buckwheat (Fagopyrum tataricum), a dicotyledonous plant belonging to the genus Polygonaceae in Fagopyrum Mill (Fagopyrum), is mainly cultivated in the provinces in the south of the Yangtze River in China, including Sichuan, Guizhou, and Shanxi (Luthar et al., 2021). Tartary buckwheat products, such as tea and noodles, are popular among consumers. It is a plant with both medicinal and edible properties. It has many health benefits, including antioxidant, anti-inflammatory, and antidiabetic effects which are closely related to its bioactive substances. A recent study has shown the αd-glucosidase inhibitory and antidiabetic activity of Tartary buckwheat polysaccharide (TBP) (Zou et al., 2021). Although few studies have documented TBP separation, purification, and structural identification (Wang et al., 2016), the dissection of different TBP components, which may exert the probiotic effect and regulate the GM composition, is not reported to date.

| Extraction of TBP
The Tartary buckwheat was washed and dried. Afterward, 10 times the volume of distilled water was added. The mixture was subjected to boiling water extraction for 3 h, which was repeated three times.
The impurities of the supernatant liquor were removed using a filter.
The extracts were then combined and concentrated. Protein was removed using the Sevag reagent (chloroform/1-Butanol, v/v = 4:1) followed by centrifugation, and anhydrous ethanol (4-volume) was added. The resulting solution was stirred and incubated overnight for protein precipitation. The precipitate was obtained by filtration using the Buchner funnel. Furthermore, the precipitate was redissolved in distilled water (60°C water bath to evaporate the ethanol). The ethanol-free precipitate was subjected to lyophilization using a freeze-drying machine (FD-2, BoYikang Experimental Instrument Co., Ltd.) to obtain TBP.

| Isolation and purification of TBP
The TBP was dissolved in the distilled water and centrifuged.
The supernatant was used for the subsequent experiment. The four elution solvents used in this study were water, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl. The phenol-sulfuric acid method was adopted and the scatter plot was constructed .
Based on the peak shape, the results were collected respectively.
After concentrating the relevant components, dialysis using 3500 Da dialysis bags (Sigma Chemical Company) and freezedrying were done. The highest content fraction was weighed, dissolved in the mobile phase, and then centrifuged. The resulting supernatant was further purified using a GPC Autopurifier system (BRT-GS) procured from the Bo Rui Saccharide Biotech Co., Ltd. and collected by the online detection combined with a refractive index detector (RI-502, SHODEX) to collect the symmetric peaks.
The collection solution was concentrated using a rotary evaporator and freeze-dried.

| TBP structure identification
The molecular weight was determined using high-performance gel permeation chromatography (HPGPC). The samples and standard solution were prepared and filtered through a 0.22μm microporous membrane. Subsequently, the solution was transferred to the injection vial. The chromatographic conditions were adjusted as follows: chromatographic column BRT105-104-102 was in tandem with gel column (8 × 300 mm), the mobile phase was 0.05 M sodium chloride solution, the flow rate was 0.6 ml/min, the column temperature was 40°C, the injection volume was 20 μl, and the detector was a RI-10A of the refractive index detector.
Monosaccharide composition was determined using an ion chromatography (IC). The solution of each monosaccharide was prepared as a 5 mg/L mixed standard. The sample was weighed in an ampoule, 3 M trifluoroacetic acid (TFA) was added, and hydrolyzed at 120°C for 3 h. The acid hydrolyzed solution was transferred to a tube and freeze-dried with liquid nitrogen. Then, 5 ml of water was added to it and vortexed. In a separate tube, 100 μl of the suspension was aspirated and 900 μl of deionized water was added followed by centrifugation. The resulting supernatant was then analyzed using IC. The chromatographic conditions were adjusted as follows: column Dionex Carbopac™ PA20 (3 × 150); mobile phase A: H 2 O; B: 15 mM sodium hydroxide; C: 15 mM sodium hydroxide and 100 mM sodium acetate; flow rate: 0.3 ml/min; injection volume: 5 μl; column temperature: 30°C; and detector: electrochemical detect.

| Effect of different fractions of polysaccharide on the growth of probiotics
Three Bifidobacteria (B. longum, B. breve, and B. adolescentis) and LGG were incubated in the lactic acid bacteria culture (MRS) medium for 2-3 days at 37°C for activation under anaerobic or aerobic conditions, respectively. The incubation was followed by three repeated passages before being prepared for use. The growth of the bacterial strains on different carbon source-containing media was measured with an enzyme-labeled instrument (Salli et al., 2021). Briefly, 20 μl of each polysaccharide fraction (2%) or glucose (2%) solution was added to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, followed by the addition of 180 μl of cell suspension containing the microorganism to be measured (1%, v/v). The final concentration of carbon source in each well was kept as 0.2% (w/v).
Glucose was used as a nonselective positive control substrate. In addition, a medium without any added carbohydrate was used as a negative control. Bifidobacterium and LGG were incubated for 36 h at 37°C under anaerobic and aerobic conditions, respectively, and optical density (OD) was measured at 600 nm for every 4 h.
The plates were shaken for 10 s before the measurements. Each bacterial-carbohydrate combination was analyzed in at least two independent experiments, each in three replicates.
Fresh stool samples were provided by three healthy volunteers (18-25 years old) who had not taken any antibiotics in the past 3 months. An autoclave was used to sterilize the stool for 20 min at 121°C before use. Stool samples from each donor were mixed in equal amounts and immediately homogenized in sterile phosphatebuffered saline (0.1 M, pH 7.2) for 1 min to obtain a stool mixture (10% w/v). The final samples were collected after filtration through four layers of sterile gauze. The filtrate was immediately stored in an anaerobic tank.
Next, 1.0 ml of 10% fecal filtrate was added to 9.0 ml of the abovementioned medium as a normal control group (group N), while the same volume of the fecal filtrate was added to 9.0 ml of medium containing 0.1 g of TBP as a TBP group (group T) (to simulate the impact of ingestion of TBP on the growth of GM). The abovementioned procedure was followed by anaerobic fermentation at 37°C.

| Changes in the SCFA during fermentation
The method described by Liu et al. (Liu et al., 2022) was used to determine the SCFAs content. Briefly, the fermentation broth was centrifuged and added to the anhydrous ethanol in a 1:1 ratio, vortexed, mixed, and centrifuged. The supernatant was used for gas chromatography-mass spectrometry (GC-MS) analysis. The peak area was recorded, and the corresponding SCFA concentration was calculated using the standard curve. The chromatographic conditions were as follows: chromatographic column: Agilent DB-WAX and capillary column (30 m × 0.25 mm ID × 0.25 μm); injection volume: 1 μl; inlet temperature: 250°C; ion source temperature: 230°C; transmission line temperature: 250°C; and quadrupole temperature: 150°C. The starting temperature of the programmed ramp-up was 90°C; it was ramped up to 180°C at a speed of 10°C/ min and maintained for 2 min and then ramped up to 250°C at 25°C/ min and maintained for 2 min. Nitrogen was used as the carrier gas, with a flow rate of 1.0 ml/min. The MS conditions were as follows: electron impact ionization source, full scan, SIM scan mode, and electron energy of 70 eV.

| Statistical analysis
All the experiments were performed in triplicates and the results were expressed as mean ± standard deviation. One-way analysis of variance was performed using IBM SPSS Statistics for Windows, version 26 (IBM Corp.). Significant differences between the groups were determined by Duncan's post hoc test. The p values <.05 were considered statistically significant. Plots were constructed using Origin 2018 (OriginLab Corporation) and GraphPad Prism version 9 for Windows (GraphPad Software).

| Isolation and purification of TBP
Tartary buckwheat has various health promoting effects due to its unique bioactive components, such as flavonoids, phenolic acids, triterpenes, and active polysaccharides (Zou et al., 2021).

| Identification of TBP structure
TBP-W, TBP-0.2, and TBP-0.5A were mainly composed of polysaccharides with two relative molecular masses. The molecular weight

| Effect of TBP on the composition of GM
GM provides complementary genetic resources for energy acquisition, production of essential vitamins, gut maturation, and immune system development to the host (Shin et al., 2015). Dietary intervention is among the main approaches leading to individual microbiota variation (Rothschild et al., 2018)  The relative abundance of microbiota at the phylum level is shown in Figure 2a. The proteobacteria in group T were significantly less than those in group N (p < .05), while the bacteroidetes were markedly higher than those of group N (p < .05). The addition of TBP causes a decrease in the normal composition of proteobacteria, as well as an increase in the bacteroidetes and firmicutes.
According to the existing studies, proteobacteria are the marker of intestinal microdysbiosis, including various pathogenic bacteria, such as Escherichia coli, Salmonella, and Shigella. An increase in the proteobacteria can lead to nutritional and metabolic disorders, and immune dysregulation in the host (Shin et al., 2015). Therefore, its reduction might be beneficial for host health. Next, the bacteroidetes belong to the major beneficial microbiota that is involved in the fermentation and utilization of polysaccharides to produce SCFAs, especially acetate and propionate (Patnode et al., 2019). The production of SCFAs is dependent on a plethora of discrete polysaccharide utilization loci that are selectively activated to facilitate glycan capture at the cell surface (Foley et al., 2016). Polysaccharides serve as the main source of energy for bacteroidaceae. The bacteroidetes can degrade various plant polysaccharides, thus increasing their relative abundance (Tamura et al., 2017). The previous finding justifies the higher relative abundance of bacteroidetes in group T as compared to group N. Among the other two major phyla, the firmicutes contain a variety of beneficial bacteria, such as Lactobacillus, and pathogenic bacteria exerting more complex health effects. In addition, actinobacterium is a common probiotic represented by  (Nagao-Kitamoto et al., 2020). Notably, the relative abundance of Shigella genera was significantly increased in the TBP group, which is consistent with the findings by Wu et al. (Wu et al., 2021). The possible reason could be the potential of Shigella to utilize a lowmolecular-weight carbon source supporting its growth in the group T. In TBP in vitro fermentation experiments, the relative abundance of Parabacteroides distasonis was markedly reduced in the group T relative to the control group (p < .05). It has been found that higher levels of P. distasonis are associated with more pronounced memory deficits (Noble et al., 2021). Generally, TBP promotes the growth of beneficial bacteria and inhibits harmful bacteria. However, a simultaneous increase in the growth of harmful bacteria Shigella genera cannot be ignored. Therefore, in-depth studies on the effect of polysaccharides on the structure of the GM are required as a future goal. To further compare the species composition differences among samples and demonstrate species abundance distribution trend for each sample, a heat map was generated for the composition analysis of different species. Figure 2c reveals the abundance data of the top 50 genera in terms of mean abundance in groups T and N. In both groups, the relative abundance of beneficial genera, including

| Effect of TBP on the composition of SCFAs
SCFAs are the main metabolites produced after fermentation and play a crucial role in immunity, inflammation, and metabolism (Yao et al., 2022). The concentration of SCFAs is often used as an indicator to determine the fermentability of polysaccharides. In this study, GC/MS was used to determine the changes in the SCFAs composition during the fermentation process. Given that the levels of caproic, valeric, isobutyric, and isovaleric acids were low and remained significantly unchanged during fermentation, only three major SCFAs, namely acetic acid, propionic acid, and butyric acid, were analyzed. GM can utilize TBP to produce SCFAs (Table 3). Notably, the experimental group with added TBP had significantly higher levels of acetic acid, propionic acid, and butyric acid than the control group during 0-48 h fermentation. Acetic acid increased from 18.17 to 65.88 μg/ml, while the level of propionic acid enhanced from 11.78 to 38.74 μg/ml. These results are in agreement with the previous studies on the fermentation of okra polysaccharides among other polysaccharides in the fecal flora . Acetic acid has been shown as a source of energy for the GM (with the most abundant SCFA in the peripheral circulation) to cross the blood-brain barrier.
It is metabolized in the muscle, kidney, heart, and brain to provide energy (Oliveira et al., 2012). Bartolomaeus et al. (2018) reported that propionic acid prevented the damage of target organs in hypertensive and atherosclerotic mice. The mechanism was mediated by maintaining the immune homeostasis against hypertension-induced cardiovascular function impairment, thereby playing a crucial role in cardiovascular health. In particular, butyric acid is useful in improving health. It has multiple physiological effects, such as maintaining intestinal homeostasis by regulating synaptopodin (SYNPO) expression. Therefore, butyric acid promotes intestinal barrier function and accelerates the repair of intestinal epithelial cell damage . Taken together, TBP can be degraded and utilized by the GM during in vitro fermentation to produce a variety of SCFAs that are beneficial to human health.

| CON CLUS ION
The current study revealed that four different structural polysaccharide fractions of TBP efficiently affected the proliferation of specific probiotic bacteria (B. longum, B. breve, B. adolescentis, and LGG). The growth of microbiota was found to be associated with the molecular weight and monosaccharide composition of different fractions. We observed that the TBP was significantly degraded and utilized by the human GM. As a result, it increased the production of beneficial microorganisms and inhibited the growth of harmful microorganisms.
Furthermore, it promoted the production of acetic acid, propionic acid, and butyric acid. We postulate that TBP can change the composition and abundance of human GM, improve the intestinal microenvironment, and ultimately offer several benefits to human health.
Taken together, the current study suggests that TBP has probiotic properties and can reshape the GM composition to exert physiological effects. Our findings further suggest that the intake of different sources of plant polysaccharides may be important in modulating intestinal microecology. Note: T, the experimental group (TBP supplement); N, the control group (no additional carbon source supplement). The results are expressed as the mean ± standard deviation, and different letters in the same column are significant (p < .05).

TA B L E 3
The concentration of shortchain fatty acids (SCFAs) at different time points of fermentation